In order to elucidate the mechanism of regulation of insulin secretion by cAMP, a systematic study of cAMP-dependent phosphoproteins will be carried out on isolated cells of the transplantable insulinoma of the Syrian hamster. Phosphoproteins labeled with 32P in isolated cells and in cells maintained in short-term tissue culture will be analyzed by two-dimensional electrophoresis and autoradiography. The influence of hormonal and pharmacologic agents that alter the insulin secretory response will be studied with respect to the kinetics of phosphorylation and dephosphorylation and the intracellular turnover of these proteins, using double isotope labeling techniques. The studies will be extended to pancreatic islets and cultured endocrine and non-endocrine cells, in order to identify those phosphoprotein(s) that may be unique to endocrine, and perhaps, insulin secreting cells. Preparations of cellular organelles will also be made on the insulinoma cells to determine the subcellular localization of the phosphoproteins and the enzymes which regulate phosphate turnover in these proteins, i.e., protein kinase and phosphoprotein phosphatase. In order to further elucidate the mechanism of cAMP-mediated insulin secretion, studies will then be directed towards the purification and characterization of selected phosphoproteins that may be involved in the intracellular translocation of the secretory granule.